Novel bacteriophage and therapeutic agent for bacterial endophthalmitis

ABSTRACT

The objective of the present invention is to provide a novel bacteriophage useful for treating bacterial endophthalmitis and a therapeutic agent for bacterial endophthalmitis comprising the novel bacteriophage. The therapeutic agent for bacterial endophthalmitis according to the present invention is characterized in comprising 1 or more bacteriophages selected from the group essentially consisting of Myoviridae Spounavirinae phiEF7H (accession number: NITE BP-02886), Myoviridae Spounavirinae phiEF19G (accession number: NITE BP-02887), Myoviridae Spounavirinae phiEF14H1 (accession number: NITE BP-02888), and mutants thereof.

TECHNICAL FIELD

The present invention relates to a novel bacteriophage useful for treating bacterial endophthalmitis and a therapeutic agent for bacterial endophthalmitis comprising the novel bacteriophage.

BACKGROUND ART

Uveitis is a disease that causes inflammation in the eyes, and endophthalmitis is a kind of uveitis. Infectious endophthalmitis among endophthalmitis is caused by bacteria, fungus, virus, parasites or the like. In particular, bacterial endophthalmitis after an ophthalmic. surgery is a social problem, since such bacterial endophthalmitis is iatrogenic. In addition, postoperative bacterial endophthalmitis quickly develops in many cases and may possibly cause blindness in a few days. If there remains corneal opacity or scarring of retina due to bacterial infection, visual function does not recover even after bacterial eradication, since eye tissue plays a role as an optical system. It is therefore very important to rapidly eliminate bacteria the germs and minimize eye tissue destruction for treating bacterial endophthalmitis.

Bacterial infection has been mainly treated by drug therapy using antibiotics since penicillin was discovered. For example, Patent document 1 discloses a method for treating endophthalmitis by using fluoroquinolone, which is a kind of synthetic antibiotics. But drug-resistant bacteria began to emerge from the 1980s due to abuse of antibiotics, and new development of antibiotics and emergence of drug-resistant bacteria have alternately appeared. There is an urgent need to develop a therapeutic agent having a mechanism different from conventional antibiotics in the present day, since multidrug-resistant organisms showing high resistance to many drugs have emerged and there are some cases difficult to be treated but the number of newly developed antibiotics has been decreased year by year.

A phage therapy using a bacteriophage has been known as a method for treating bacterial infection without using antibiotics. Since a bacteriophage has higher specificity than antibiotics and does not exhibit a bacteriolytic action to bacteria other than a specific bacterium in many cases, it is possible in theory to select a bacteriophage to be used which bacteriophage is harmless to not only a patient but also the other bacteria which is not pathogenic and which prevents opportunistic infection disease, such as good bacteria in intestinal flora. Thus, a phage therapy is hardly considered to cause an adverse reaction. In addition, a dosage amount and a frequency of administration may be small, since a bacteriophage infects a host bacterium to grow proliferously.

A phage therapy was clinically applied mainly in Eastern Europe and Russia. In recent years, a phage therapy has attracted attention as a therapeutic measure against drug-resistant bacteria also in Western European countries. The possibility of a phage therapy is specified in national action plans relating drug resistance in Japan.

PRIOR ART DOCUMENT Patent Document

Patent Document 1: JP 2010-536860 T

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

As described above, if bacteria germs cannot be rapidly eliminated in bacterial endophthalmitis, a destruction and a scarring in an eye tissue progress and visual functions cannot be expected to be recovered. A phage is the only therapeutic agent that increases in a lesion of disease, since a phage infects a bacterium to grow proliferously. Also, a phage is considered to have low toxicity due to high host specificity. In addition, a phage may effectively inhibit a destruction of an eye tissue, since a phage can kill a bacterium more rapidly than an antimicrobial agent. Furthermore, a phage can be expected to be effective against drug-resistant bacteria, since the mechanism of action is different between a phage and an antimicrobial agent.

Accordingly, the objective of the present invention is to provide a novel bacteriophage useful for treating bacterial endophthalmitis and a therapeutic agent for bacterial endophthalmitis comprising the novel bacteriophage.

Means for Solving the Problems

The inventors of the present invention conducted extensive studies to solve the above problems. As a result, the inventors completed the present invention by finding novel bacteriophages which exhibit a bacteriolytic action to a plurality of enterococcus strains derived from bacterial endophthalmitis. The enterococcus is one of four major responsible bacteria to cause bacterial endophthalmitis.

Hereinafter, the present invention is described.

[1] Myoviridae Spounavirinae phiEF7H (accession number: NITE BP-02886).

[2] Myoviridae Spounavirinae phiEF19G (accession number: NITE BP-02887).

[3] Myoviridae Spounavirinae phiEF14H1 (accession number: NITE BT-02888).

[4] A therapeutic agent for bacterial endophthalmitis, comprising 1 or more bacteriophages selected from the group essentially consisting of Myoviridae Spounavirinae phiEF7H (accession number: NITE BP-02886), Myoviridae Spounavirinae phiEF19G (accession number: NITE BP-02887), Myoviridae Spounavirinae phiEF14H1 (accession number: NITE BP-02888), and mutants thereof.

[5] The therapeutic agent for bacterial endophthalmitis according to the above [4], wherein the mutant comprises any one of the following nucleotide sequences:

-   -   (1) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ         ID NO: 3 having deletion, substitution and/or addition of 1 or         more and 1500 or less of bases, wherein the mutant having the         nucleotide sequence exhibits a bacteriolytic action against a         bacterium causing bacterial endophthalmitis;     -   (2) a nucleotide sequence having a sequence identity of 99.9% or         more to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the         mutant having the nucleotide sequence exhibits a bacteriolytic         action against a bacterium causing bacterial endophthalmitis.

[6] The therapeutic agent for bacterial endophthalmitis according to the above [4] or [5], further comprising 1 or more bacteriophages selected from the group essentially consisting of Myoviridae Spounavirinae phiEF24C, Myoviridae Spounavirinae phiEF24C-P2, and mutants thereof.

[7] The therapeutic agent for bacterial endophthalmitis according to any one of the above [4] to [6], wherein the therapeutic agent is a liquid medicine.

Effect of the Invention

A general bacteriophage has very high specificity and exhibits a bacteriolytic action against only one strain in many cases. On the one hand, a phage therapy for bacterial endophthalmitis caused by various strains of bacteria becomes possible according to the present invention. Thus, the present invention is very useful as a therapeutic measure for endophthalmitis without using antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph to show each clinical score of endophthalmitis model mice to which bacteriophage phiEF7H according to the present invention was administered.

FIG. 2 is a graph to show each clinical score of endophthalmitis model mice to which bacteriophage phiEF19G according to the present invention was administered.

FIG. 3 is a graph to show each clinical score of endophthalmitis model mice to Which bacteriophage phiEF14H1 according to the present invention was administered.

FIG. 4 is a graph to show the numbers of viable enterococci in the eye of endophthalmitis model mice to which bacteriophage phiEF7H according to the present invention was administered.

FIG. 5 is a graph to show the numbers of viable enterococci in the eye of endophthalmitis model mice to which bacteriophage phiEF19G according to the present invention was administered.

FIG. 6 is a graph to show the numbers of viable enterococci in the eye of endophthalmitis model mice to which bacteriophage phiEF14H1 according to the present invention was administered.

FIG. 7 is a graph to show a myeloperoxidase activity in the eye of endophthalmitis model mice to which bacteriophage phiEF7H according to the present invention was administered.

FIG. 8 is a graph to show a myeloperoxidase activity in the eye of endophthalmitis model mice to which bacteriophage phiEF19C7 according to the present invention was administered.

FIG. 9 is a graph to show a myeloperoxidase activity in the eye of endophthalmitis model mice to which bacteriophage phiEF14H1 according to the present invention was administered.

MODE FOR CARRYING OUT THE INVENTION

The bacteriophage Myoviridae Spounavirinae phiEF7H, hereinafter abbreviated as “phiEF7H strain”, of the present invention has been internationally deposited to Authority Depository as follows. The genome nucleotide sequence of the phiEF7H strain is shown as SEQ ID NO: 1.

-   -   (i) Name and address of Authority Depository     -   Name: NITE Patent Microorganisms Depositary (NPMD), Biological         Resource Center, National Institute of Technology and Evaluation     -   Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba         2920818, Japan     -   (ii) Date of the original deposit: Feb. 14, 2019     -   (iii) Accession number: NITE BP-02886

The bacteriophage Myoviridae Spounavirinae phiEF19G, hereinafter abbreviated as “phiEF19G strain”, of the present invention has been internationally deposited to Authority Depository as follows. The genome nucleotide sequence of the phiEF19G strain is shown as SEQ ID NO: 2.

-   -   Name and address of Authority Depository     -   Name: NITE Patent Microorganisms Depositary (NPMD), Biological         Resource Center, National Institute of Technology and Evaluation     -   Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba         2920818, Japan     -   (ii) Date of the original deposit: Feb. 14, 2019     -   (iii) Accession number NITE BP-02887

The bacteriophage Myoviridae Spounavirinae phiEF14H1, hereinafter abbreviated as “phiEF14H1 strain”, of the present invention has been internationally deposited to Authority Depository as follows. The genome nucleotide sequence of the phiEF14H1 strain is shown as SEQ ID NO: 3

-   -   (i) Name and address of Authority Depository     -   Name NITE Patent Microorganisms Depositary (NPMD), Biological         Resource Center, National Institute of Technology and Evaluation     -   Address: #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba         2920818, Japan     -   (ii) Date of the original deposit: Feb. 14, 2019     -   (iii) Accession number: NITE BP-02888

Myoviridae Spounavirinae phiEF7H, Myoviridae Spounavirinae phiEF19G, and Myoviridae Spounavirinae phiEF14H1 respectively means phiEF7H, phiEF19G, and phiEF14H1 belonging to Myoviridae Spounavirinae. The description of the other phage strain has the same meaning.

Myoviridae Spounavirinae phiEF24C, hereinafter abbreviated as “phiEF24C strain”, is a publically known bacteriophage and described in, for example, Uchiyama J., et al., FEMS Microbiol Lett., 2008, 278(2), pp. 200-206.

Myoviridae Spounavirinae phiEF24C-P2, hereinafter abbreviated as “phiEF24C-P2 strain”, is a publically known bacteriophage and for example, the accession number thereof is AB609718.

The therapeutic agent for bacterial endophthalmitis according to the present invention comprises 1 or more bacteriophages selected from the group essentially consisting of the phiEF7H strain, the phiEF19G strain, the phiEF14H1 strain, and the mutants thereof. Hereinafter, the above bacteriophages are conveniently described as “bacteriophage 1” in some cases.

The genome of the above-described mutant corresponds to the genome of the phiEF7H strain, the phiEF19G strain or the phiEF14H1 strain having one or more mutations and exhibits the bacteriolytic action against a bacterium responsible for bacterial endophthalmitis. The bacterium responsible for bacterial endophthalmitis is not particularly restricted and is exemplified by enterococcus such as Enterococcus faecalis.

For example, the above-described mutant has the genome having any one of the following nucleotide sequences.

(1) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 having 1 or more and 1500 or less of deletion, substitution and/or addition, wherein the mutant having the nucleotide sequence exhibits a bacteriolytic action against a bacterium causing bacterial endophthalmitis;

(2) a nucleotide sequence having an identity of 99.9% or more With the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the mutant having the nucleotide sequence exhibits a bacteriolytic action against a bacterium causing bacterial endophthalmitis.

The number of the mutation in the above-described nucleotide sequence (1) is preferably 1200 or less, more preferably 1000 or less, and even more preferably 800 or less, 500 or less, 200 or less, or 100 or less.

The identity in the above-described nucleotide sequence (2) is preferably 99.92% or more, more preferably 99.95% or more, and even more preferably 99.98% or more, or 99.99% or more.

A person skilled in the art can determine an identity between nucleotide sequences by an ordinary method such as a method using Clustal (http://www.clustal.org/omega/), which is a program for multiple alignment of amino acid sequence. In addition, a position of the mutation in the above-described nucleotide sequences (1) and (2) can be similarly determined by an ordinary method.

It can be determined by an ordinary method whether a mutant exhibits a bacteriolytic action against a bacterium causing bacterial endophthalmitis or not. For example, a bacterium is isolated from an affected area of a bacterial endophthalmitis patient, the bacterium is cultivated, a mutant is added to the culture medium, the bacterium is further cultivated, and then the bacterium is observed. The bacteriolytic action may be evaluated depending on a change of bacterial morphology and an existence or non-existence of plaque. Also, the bacteriolytic action of the phiEF7H strain, the phiEF19G strain and the phiEF14H1 strain according to the present invention, and the phiEF24C strain and the phiEF24C-P2 strain against a bacterium causing bacterial endophthalmitis may be preliminarily tested in a similar manner.

The therapeutic agent for bacterial endophthalmitis according to the present invention may comprise the 1 or more bacteriophages 2 selected from the group essentially consisting of the phiEF24C strain, the phiEF24C-P2 strain, and the mutants thereof in addition to the bacteriophage 1 selected from the group essentially consisting of the phiEF7H strain, the phiEF19G strain, the phiEF14H1 strain, and the mutants thereof. The bacteriophage 2 may supplementally exhibit a bacteriolytic action against a bacterium causing bacterial endophthalmitis to which the bacteriophage 1 does not exhibit a bacteriolytic action.

The therapeutic agent for bacterial endophthalmitis may be orally administered but is preferably administered through an intravenous drip infusion into the blood or by injection into the eye, since bacterial endophthalmitis must be immediately treated.

The therapeutic agent for bacterial endophthalmitis is produced from a suspension of the above-described bacteriophage. Such a suspension of the bacteriophage can be prepared by an ordinary method. For example, a bacterium to which the objective bacteriophage exhibits a bacteriolytic action is cultivated in a liquid culture medium, and then the objective bacteriophage is added to the culture medium in order to transmit the bacteriophage to the bacterium. Next, the bacterium is subjected to lysis by continuing the cultivation. As a result, the bacteriophage is increased in the culture medium. After a concentration of the bacteriophage in the culture medium becomes appropriate, for example, becomes included in the range of 10² phages/mL or more and 10¹² phages/mL or less, a solid component other than the bacteriophage is removed by centrifugation, filtration or the like to obtain a suspension of the bacteriophage.

A stabilizing agent is preferably added to the above liquid culture medium and the suspension to stably preserve the bacteriophage. An example of such a stabilizing agent includes a pH adjuster and a buffer to maintain the pH in the range of 6 or more and 8 or less, preferably 6.5 or more and 7.5 or less; an amino acid such as glycine, arginine and lysine; and a salt such as sodium chloride and calcium chloride.

A final concentration of the bacteriophage in the liquid therapeutic agent for bacterial endophthalmitis is preferably 10³ pfu/mL or more and 10¹⁴ pfu/mL or less. Also, the liquid therapeutic agent for bacterial endophthalmitis is preferably isotonic or nearly isotonic with a body fluid such as blood. In addition, the solid therapeutic agent for bacterial endophthalmitis for oral administration can be produced by, for example, adding an excipient or the like to the above bacteriophage suspension to be granulated.

A dosage amount of the therapeutic agent for bacterial endophthalmitis according to the present invention is not particularly restricted and may be appropriately adjusted depending on age, sex, weight, symptomatic state, severity or the like of a patient. For example, the titer of the bacteriophage 1 to be administered to a human or an animal per one time can be adjusted to about 10² pfu or more. and about 10⁸ pfu or less. A frequency of administration per one day can be adjusted to about 0.5 or more and about 6 or less.

The present application claims the benefit of the priority date of Japanese patent application No. 2018-44407 filed on. Mar. 12, 2018. All of the contents of the Japanese patent application No. 2018-44407 filed on Mar. 12, 2018, are incorporated by reference herein.

EXAMPLES

Hereinafter, the examples are described to demonstrate the present invention more specifically, but the present invention is in no way restricted by the examples, and the examples can be appropriately modified to be carried out within a range which adapts to the contents of this specification. Such a modified example is also included in the range of the present invention.

Example 1: In Vitro Experiment

Each strain of Enterococcus faecalis causing bacterial endophthalmitis was cultivated overnight. An agar was added to Tryptic Soy Broth manufactured by Bacto in a concentration of 0.5% to prepare a soft agar. The temperature of the soft agar was maintained to be 55° C. On a Tryptic Soy Broth agar medium containing 1.5% agar, 0.2 mL of a preliminary liquid culture was inoculated and the soft agar was further poured to be mixed. The double layered agar medium was left at room temperature until the upper soft agar became solidified.

Streaks of one loop amount of the each bacteriophage liquid were drawn on the double layered agar medium using a 10 μL inoculation loop and subjected to cultivation at 37° C. overnight. Then, Whether plaque was formed or not and the morphology of the bacteria were evaluated. The result was shown in Tables 1 and 2. In Tables 1 and 2, “Excellent” demonstrated that plaque was clearly formed, “Good” demonstrates that plaque was not formed but bacteriolysis was slightly observed, and “Poor” demonstrates that bacteriolysis was not observed.

TABLE 1 E. faecalis strain phiEF7H phiEF17H phiEF18H phiEF19G phiEF28H phiM1EF28 933-1 Excellent Excellent Good Excellent Excellent Poor 1505063 Excellent Excellent Excellent Excellent Excellent Good 11. M-IOL Excellent Good Excellent Excellent Excellent Poor 12. M-AC Excellent Poor Poor Excellent Good Poor 15. K-EO Excellent Excellent Excellent Excellent Excellent Poor

TABLE 2 E. faecalis strain phiEF24C phiEF24C-P2 phiM1EF2 phiM2EF28 phiEF14H1 933-1 Good Good Good Poor Excellent 1505063 Good Good Excellent Excellent Excellent 11. M-IOL Excellent Excellent Poor Poor Excellent 12. M-AC Poor Poor Poor Poor Excellent 15. K-EO Poor Poor Good Poor Excellent

It was experimentally demonstrated as the result shown in Tables 1 and 2 that the bacteriophages phiEF7H, phiEF19G and phiEF14H1 exhibit the bacteriolytic action against all of tested 5 enterococci derived from endophthalmitis and broad antimicrobial spectrum against E. faecalis as one of bacteria causing endophthalmitis. Among the above-described enterococci, 11. M-IOL was isolated from the intraocular lens of an endophthalmitis patient and 12. M-AC was separated from the anterior chamber of the same patient.

Example 2: Identification of Novel Bacteriophage

The genus and species of bacteriophages phiEF7H, phiEF19G and phiEF14H1, of which effects on bacteria causing bacterial endophthalmitis were particularly demonstrated in the above-described Example 1, were determined. Specifically, the DNA of each bacteriophage was purified and the nucleotide sequence thereof was determined. The genome nucleotide sequence of phiEF7H is shown as SEQ ID NO: 1, the genome nucleotide sequence of phiEF19G is shown as SEQ ID NO: 2, and the genome nucleotide sequence of phiEF14H1 is shown as SEQ ID NO: 3.

Then, the obtained genome nucleotide sequence was analyzed using BLASTn; as a result, the genome nucleotide sequences had a high homology with that of phiEF24C-P2 as 89% of Query cover, 0.0 of E-value and 98% of Identity, but no same publically known genome nucleotide sequence was found. Since the phiEF24C-P2 is a publically known bacteriophage disclosed in Uchiyama, J., et al., 2008, Appl. Environ. Microbiol., 74, pp. 4149-4163 and belongs to Myoviridae Spounavirinae, it was clarified that phiEF7H, phiEF19G and phiEF14H1 are novel bacteriophages belonging to Myoviridae Spounavirinae.

Example 3: In Vivo Experiment

To the right eye vitreous body of four mice, 1×10³ cells of Enterococcus 933-1 strain, 1505063 strain, 11. M-IOL and 15. K-EO strain separated from an endophthalmitis patient, and vancomycin resistant enterococcus VRE2 were respectively administered to induce endophthalmitis. Phages phiEF7H, phiEF19G or phiEF14H1, or normal saline solution as control was administered to the vitreous body 6 hours after the infection. Clinical score was evaluated 24 hours after the infection, and then the eye was enucleated to count the number of the viable enterococcus and evaluate myeloperoxidase (MPO) activity to estimate the number of inflammatory cell infiltrate. The average value of clinical score is shown in FIGS. 1 to 3, the average value of the number of viable enterococcus is shown in FIGS. 4 to 6, and the average value of MPO activity is shown in FIGS. 7 to 9. The clinical score of endophthalmitis was determined in accordance with the following criteria. In FIGS. 1 to 9, * demonstrates that there is a significant difference (p<0.05, independent 2 group Student's t-test), and ** demonstrates that there is a significant difference (p<0.01).

Score 0: anterior chamber and vitreous body are transparent, and clear view of the retina

Score 1: there are a mild degree of anterior chamber flare and a mild degree of opacified vitreous haze, and slightly obscured view of the retina

Score 2: there are moderately severe anterior chamber flare, dense vitreous haze, and moderately obscured view of the retina

Score 3: there are intense anterior chamber flare, opaque vitreous, and completely obscured view of the retina

Score 4: there is anterior chamber hemorrhage in addition to the findings of Score 3

In the normal saline solution-administered group, endophthalmitis was developed, the ocular fundus was not visible as a result of fibrin precipitation and bleeding in the anterior chamber, the number of viable bacteria in the eye was increased to about 1×10⁸, and MPO activity was increased 24 hours after the infection of enterococcus.

On the one hand, when the bacteriophage of the present invention was administered after the infection of enterococcus, clinical score was increased and MPO activity tended to be increased in the case where phage phiEF7H was administered against the endophthalmitis caused by enterococcus 12M-AC, but all of the clinical score, the number of viable bacteria and MPO activity tended to be reduced and often significantly reduced in the other cases as the result shown in FIGS. 1 to 9.

It is clear from the above results that all of phages phiEF7H, phiEF19G and phiEF14H1 have effect on endophthalmitis, and endophthalmitis may be treated more effectively by combining two or more of the phages. 

1-7. (canceled)
 8. Myoviridae Spounavirinae phiEF7H (accession number: NITE BP-02886).
 9. Myoviridae Spounavirinae phiEF19G (accession number: NITE BP-02887).
 10. Myoviridae Spounavirinae phiEF14H1 (accession number: NITE BP-02888).
 11. A treatment agent for bacterial endophthalmitis, comprising 1 or more bacteriophages selected from the group essentially consisting of Myoviridae Spounavirinae phiEF7H (accession number: NITE BP-02886), Myoviridae Spounavirinae phiEF19G (accession number: NITE BP-02887), Myoviridae Spounavirinae phiEF14H1 (accession number: NITE BP-02888), and mutants thereof, wherein the mutant comprises any one of the following base sequences: (1) a base sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 having deletion, substitution and/or addition of 1 or more and 1500 or less of bases, wherein the mutant having the base sequence exhibits a bacteriolytic action against a bacterium causing bacterial endophthalmitis; (2) a base sequence having a sequence identity of 99.9% or more to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, wherein the mutant having the base sequence exhibits a bacteriolytic action against a bacterium causing bacterial endophthalmitis.
 12. The treatment agent for bacterial endophthalmitis according to claim 11, wherein the number of the mutation of the deletion, substitution and/or addition is 800 or less, and the sequence identity is 99.95% or more.
 13. The treatment agent for bacterial endophthalmitis according to claim 11, further comprising 1 or more bacteriophages selected from the group essentially consisting of Myoviridae Spounavirinae phiEF24C, Myoviridae Spounavirinae phiEF24C-P2, and mutants thereof.
 14. The treatment agent for bacterial endophthalmitis according to claim 11, wherein the treatment agent is a liquid medicine. 